A. Field of the Invention
The present invention relates to telomerase compositions and methods connected therewith. Particularly disclosed are genes encoding the template RNA of telomerase in Saccharomyces cerevisiae and various telomerase-associated proteins. Methods of using such genes and other related biological components are also provided.
B. Description of the Related Art
DNA polymerases synthesize DNA in a 5xe2x80x2 to 3xe2x80x2 direction and require a primer to initiate synthesis. These restrictions pose a problem for the complete replication of linear chromosomes (Watson, 1972; Olovnikov, 1973). In the absence of a specialized mechanism to maintain terminal sequences, multiple replication cycles would cause chromosomes to progressively shorten from their ends.
Telomeres are specialized nucleoprotein complexes that constitute the ends of eukaryotic chromosomes and protect them from degradation and end-to-end fusion (Zakian, 1989; Blackburn, 1991; Price, 1991; Henderson and Larson, 1991; Wright et al., 1992; Blackburn, 1994). When telomeres are absent, the instability of non-telomeric chromosomal ends leads to chromosome loss (Sandell and Zakian, 1993). In addition, telomeres are required for the complete replication of chromosomes (Zakian, 1989; Blackburn, 1991; Price, 1991; Henderson and Larson, 1991; Wright et al., 1992; Blackburn, 1993; 1994).
In many eukaryotes, telomeres are composed of simple tandem repeats, with the 3xe2x80x2-terminal strand composed of G-rich sequences (Zakian, 1989; Blackburn, 1991; Price, 1991; Henderson and Larson, 1991; Wright et al., 1992; Blackburn, 1994). Certain insights into the mechanism by which telomeric DNA is maintained has come from the identification of telomerase activity in several species of ciliates, as well as in extracts of Xenopus, mouse, and human cells (Greider and Blackburn, 1985; 1987; 1989; Zahler and Prescott, 1988; Morin, 1989; Prowse et al., 1993; Shippen-Lentz and Blackburn, 1989; Mantell and Greider, 1994).
Telomerase is a ribonucleoprotein enzyme that elongates the G-rich strand of chromosomal termini by adding telomeric repeats (Blackburn, 1993). This elongation occurs by reverse transcription of a part of the telomerase RNA component, which contains a sequence complementary to the telomere repeat. Following telomerase-catalyzed extension of the G-rich strand, the complementary DNA strand of the telomere is presumably replicated by more conventional means.
Germline cells, whose chromosomal ends must be maintained through repeated rounds of DNA replication, do not decrease their telomere length with time, presumably due to the activity of telomerase (Allsopp et al., 1992). In contrast, somatic cells appear to lack telomerase, and their telomeres shorten with multiple cell divisions (Allsopp et al., 1992; Harley et al., 1990; Hastie et al., 1990; Lindsey et al., 1991; Vaziri et al., 1993; Counter et al., 1992; Shay et al., 1993; Klingelhutz et al., 1994; Counter et al., 1994a;b).
Telomerase is believed to have a role in the process of cell senescence (de Lange, 1994; Greider, 1994; Harley et al., 1992). The repression of telomerase activity in somatic cells is likely to be important in controlling the number of times they divide. Indeed, the length of telomeres in primary fibroblasts correlates well with the number of divisions these cells can undergo before they senescence (Allsopp et al., 1992). The loss of telomeric DNA may signal to the cell the end of its replicative potential, as part of an overall mechanism by which multicellular organisms limit the proliferation of their cells.
Due to its role in controlling replication, telomerase has also recently been implicated in oncogenesis (de Lange, 1994; Greider, 1994; Harley et al., 1992). It is thought that late stage tumors probably require the reactivation of telomerase in order to avoid total loss of their telomeres and massive destabilization of their chromosomes. Immortalized cell lines produced from virally transformed cultures have active telomerase and stable telomere lengths (Counter et al., 1992; Shay et al., 1993; Klingelhutz et al., 1994; Counter et al., 1994b). Recently, telomerase activity has also been detected in human ovarian carcinoma cells (Counter et al., 1994a).
Telomerase is thus an important component of eukaryotic cells, the dysfunction of which can have significant consequences. Although present knowledge concerning telomerase is increasing, there is a marked need for individual telomerase components to be isolated and for further analytical methods to be developed. The creation of a system for manipulating telomerase in a genetically tractable eukaryotic organism would be particularly valuable.
The present invention overcomes these and other drawbacks inherent in the prior art by providing purified telomerase components and systems for isolating further components and for developing agents with the capacity to modify telomerase actions. Particular aspects of-this invention concern the isolation and uses of several telomerase-associated genes from Saccharomyces cerevisiae, including the telomerase RNA template gene.
In certain aspects, this invention concerns nucleic acid segments that hybridize to, or that have sequences in accordance with, SEQ ID NO:1, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:19, SEQ ID NO:31 or SEQ ID NO:23. SEQ ID NO:1 represents a telomerase RNA template-encoding sequence, also termed TLC1; and each of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:19, SEQ ID NO:31 and SEQ ID NO:23 represent sequences that encode telomerase-associated polypeptides, also termed STR sequences (STR1, STR3, STR4, STR5 and STR6, respectively).
Both the gene TLC1 (SEQ ID NO:1 and the complementary sequence, SEQ ID NO:4), and the template RNA, include a CA-rich region. The CA-rich region is represented by SEQ ID NO:3. In the RNA template, the CA-rich region is reversed transcribed to synthesize the GT-rich telomeric repeats. An example of the GT-rich telomeric sequence is represented by SEQ ID NO:2.
The present invention generally concerns non-ciliate eukaryotic telomerase components. These are represented by telomerase components from mammalian cells, including human cells, and telomerase components from other non-ciliate species. One significant contribution of this invention is the development of methods of utilizing telomerase components, which methods are functional in useful eukaryotic cells. xe2x80x9cUseful eukaryotic cellsxe2x80x9d particularly include human cells, as these are directly relevant to the development of diagnostics and therapeutics for human use, and cells of genetically tractable eukaryotic organisms, as these are recognized to have significant value in scientific terms and, ultimately, in drug development. The preferred non-ciliate telomerase components of the invention are thus mammalian, drosophila and yeast telomerase components.
A. DNA Segments and Vectors
The invention thus provides nucleic acid segments that are characterized as nucleic acid segments that include a sequence region that consists of at least 17 contiguous nucleotides that have the same sequence as, or are complementary to, 17 contiguous nucleotides of SEQ ID NO:1, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:19, SEQ ID NO:31 or SEQ ID NO:23.
The nucleic acid segments of the invention are further characterized as being of from 17 to about 10,000 nucleotides in length, which nucleic acid segments hybridize to the nucleic acid segment of SEQ ID NO:1, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:19, SEQ ID NO:31 or SEQ ID NO:23, or the complement thereof, under standard hybridization conditions.
xe2x80x9cComplementaryxe2x80x9d or xe2x80x9ccomplementxe2x80x9d, in terms of nucleic acid segments that are complementary to those listed above, or that hybridize to a complement of such nucleic acid segments, means that the nucleic acid sequences are capable of base-pairing to a given sequence, such as the sequences of SEQ ID NO:1, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:19, SEQ ID NO:31 or SEQ ID NO:23, according to the standard Watson-Crick complementarity rules. That is, the larger purines will base pair with the smaller pyrimidines to form combinations of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T), in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA.
Encompassed within the nucleic acid sequences of the invention are full-length DNA sequences or other DNA segments that have a sequence region that encodes a peptide, polypeptide or protein and that may be used, for example, in recombinant expression. Also included within the nucleic acid sequences are DNA and RNA segments for use in nucleic acid hybridization embodiments, such as in cloning.
The smaller nucleic acid segments may be termed probes and primers. The individual sequences of 17, 20, 30, 50 or so nucleotide sequence stretches, for example, may be readily identified by xe2x80x9cbreaking downxe2x80x9d the longer sequences disclosed herein to provide one or more shorter sequences. Using an exemplary length of 17 bases, each of the 17-mer possibilities from the DNA sequences described herein have been defined and are listed in Table 2.
In certain embodiments, the invention provides isolated DNA segments and recombinant vectors that have one or more sequence regions that encode one or more non-ciliate eukaryotic telomerase components, and preferably, those that encode one or more yeast (S. cerevisiae) telomerase components. The creation and use of recombinant host cells, through the application of DNA technology, that express yeast or other non-ciliate eukaryotic telomerase components is also encompassed by the invention.
As used herein, the term xe2x80x9ctelomerase componentxe2x80x9d refers to a biological component that is associated with a non-ciliate eukaryotic telomerase complex, such as a mammalian, drosophila or yeast telomerase component. Preferably, the telomerase components will be associated with a yeast telomerase complex. A xe2x80x9ctelomerase complexxe2x80x9d in this sense is a ribonucleoprotein enzyme complex that functions to elongate the G-rich strand of eukaryotic, and preferably yeast, chromosomal termini by adding telomeric repeats. Telomerase components (or telomerase-associated components) therefore include both RNA and polypeptidyl components.
An important component of telomerase is the telomerase RNA template or template sequence. The term xe2x80x9ctelomerase RNA templatexe2x80x9d, as used herein, refers to a non-ciliate eukaryotic, such as a mammalian, drosophila, or preferably, a yeast telomerase RNA component that includes a sequence that is complementary to the telomere repeat, i.e., that is complementary to the G-rich or GT-rich sequences of chromosomal termini. The telomerase RNA template is thus an isolated RNA component that has a C-rich or CA-rich sequence and that, by interacting with other telomerase components, functions to extend telomeric repeats. The telomerase RNA template may also be defined as the telomerase substrate for reverse transcription.
Further telomerase components are telomerase-associated proteins and polypeptides. The xe2x80x9ctelomerase-associated proteins and polypeptidesxe2x80x9d of this invention are proteins, polypeptides or peptides that are required for telomerase function in non-ciliate eukaryotic cells, and preferably, in yeast cells. Such telomerase-associated proteins and polypeptides will generally be physically and functionally associated with the telomerase complex in the nucleus, however, they may also be proteins or polypeptides that only associate with the telomerase complex for certain periods of time, at defined points of the cell cycle, or may be present only in certain cell types of a multicellular organism.
Telomerase-associated proteins, polypeptides and peptides may have either functional or structural roles within the telomerase complex. That is, they may have a catalytic or regulatory role, or may form the scaffolding of the telomerase structure. The telomerase-associated proteins or polypeptides may function only in terms of telomerase activity, i.e., they may be telomerase-restricted; or they may have other biological functions within the cell nucleus, such as in other aspects of chromosome replication and stability, or may even have cytoplasmic functions.
The telomerase DNA segments of the present invention are thus DNA segments isolatable from non-ciliate eukaryotic cells, and preferably, from yeast cells, that are free from total genomic DNA and that include a sequence region that is capable of expressing a telomerase RNA or polypeptide component. The DNA segments may, in certain embodiments, also be defined as those capable of inhibiting the telomerase activity of a cell by over-expression in a cell that previously contained telomerase activity. In further embodiments, the DNA segments may be defined as those capable of conferring telomerase activity to a host cell when incorporated into a cell that has been rendered deficient in such activity.
As used herein, the term xe2x80x9cDNA segmentxe2x80x9d refers to a DNA molecule that has been isolated free of total genomic DNA of a particular species, such as a mammal, drosophila or yeast species. Therefore, a DNA segment that comprises a sequence region that encodes a telomerase-associated component refers to a DNA segment that includes telomerase-associated component coding sequences or regions, yet is isolated away from, or purified free from, total genomic DNA of the species from which the DNA segment is obtained. Included within the term xe2x80x9cDNA segmentxe2x80x9d, are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like.
Similarly, a telomerase-associated gene is a DNA segment comprising an isolated or purified gene that includes a sequence region that encodes a component associated with a mammalian, drosophila, or preferably, with a yeast telomerase. The term xe2x80x9can isolated gene associated with a non-ciliate eukaryotic telomerasexe2x80x9d, as used herein, refers to a DNA segment including telomerase RNA or protein coding sequences or regions and, in certain aspects, regulatory sequences, isolated substantially away from other naturally occurring genes or encoding sequences. In this respect, the term xe2x80x9cgenexe2x80x9d is used for simplicity to refer to a functional RNA, protein, polypeptide or peptide encoding unit or region. As will be understood by those in the art, this functional term includes both genomic sequences, cDNA sequences and smaller engineered gene segments that express, or may be adapted to express, RNA molecules, proteins, polypeptides or peptides.
xe2x80x9cIsolated substantially away from other coding sequencesxe2x80x9d means that the gene of interest, in this case a telomerase-associated gene, forms the significant part of the sequence or coding region of the DNA segment, and that the DNA segment does not contain large portions of naturally-occurring coding DNA, such as large chromosomal fragments or other functional genes or cDNA coding regions. Of course, this refers to the DNA segment as originally isolated, and does not exclude genes or coding regions later added to the segment by the hand of man.
In particular embodiments, the invention concerns isolated DNA segments and recombinant vectors incorporating DNA sequences that include an isolated gene or sequence region that encodes a non-ciliate eukaryotic telomerase RNA template, such as a mammalian, drosophila, or preferably, a yeast telomerase RNA template. This aspect of the invention is exemplified by DNA segments and genes that encode the S. cerevisiae telomerase RNA template sequence of CACCACACCCACACAC (SEQ ID NO:3).
A variety of oligonucleotides, DNA segments and genes that encode the CACCACACCCACACAC (SEQ ID NO:3) telomerase RNA template sequence are made possible by the discovery of the present inventors"". These include sequences from SEQ ID NO:1, and the complementary strand, SEQ ID NO:4. The sequence from SEQ ID NO:1 that includes the template-encoding region of CACCACACCCACACAC (SEQ ID NO:3) is particularly represented by the contiguous DNA sequence from position 468 to position 483 of SEQ ID NO:1. Such DNA segments will have a minimum length of 17 nucleotides, and are exemplified by the contiguous DNA sequences from position 467 to position 483, or from position 468 to position 484, of SEQ ID NO:1.
DNA segments longer than 17 bases are also contemplated, in increments of single integers up to and including the 1301 bases of SEQ ID NO:1, and even longer. The contiguous sequences from SEQ ID NO:1 may be equidistant around the template-encoding region of SEQ ID NO:3, or they may have the SEQ ID NO:3 region located substantially towards the beginning or towards the end of the given sequence. DNA segments may thus have sequences in accordance with the contiguous sequences between about position 450 or 460 and about position 485 of SEQ ID NO:1; between about position 300 or 400 and about position 500, 600 or 700 of SEQ ID No:1; between about position 100 or 200 and about position 800, 900, 1000, 1100 or 1200 of SEQ ID NO:1; or between any of the afore-mentioned ranges and intermediates thereof. DNA segments and isolated genes that include the full-length DNA sequence of SEQ ID NO:1 are also contemplated.
In further embodiments, the invention provides isolated DNA segments, genes and vectors incorporating DNA sequences that encode a non-ciliate eukaryotic telomerase-associated polypeptide, such as a mammalian, drosophila or yeast, telomerase-associated polypeptide, as exemplified by yeast polypeptides that includes within their amino acid sequence a contiguous amino acid sequence from SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22 or SEQ ID NO:24.
The term xe2x80x9ca contiguous amino acid sequence from SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22 or SEQ ID NO:24xe2x80x9d means that a contiguous sequence is present that substantially corresponds to a contiguous portion of one of the afore-mentioned sequences and has relatively few amino acids that are not identical to, or a biologically functional equivalent of, the amino acids of SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22 or SEQ ID NO:24. The term xe2x80x9cbiologically functional equivalentxe2x80x9d is well understood in the art and is further defined in detail herein. Accordingly, sequences that have between about 70% and about 80%; or more preferably, between about 81% and about 90%; or even more preferably, between about 91% and about 99%; of amino acids that are identical or functionally equivalent to the amino acids of SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22 or SEQ ID NO:24 will be sequences in accordance with the present invention.
The protein-encoding DNA segments, genes and vectors may include within their sequence region a contiguous nucleic acid sequence from SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:19, SEQ ID NO:31 or SEQ ID NO:23. The term xe2x80x9ca contiguous nucleic acid sequence from SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:19, SEQ ID NO:31 or SEQ ID NO:23xe2x80x9d is used in the same sense as described above and means that the nucleic acid sequence substantially corresponds to a contiguous portion of one of the designated sequences and has relatively few codons that are not identical, or functionally equivalent, to the codons of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:19, SEQ ID NO:31 or SEQ ID NO:23. The term xe2x80x9cfunctionally equivalent codonxe2x80x9d is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine, and also refers to codons that encode biologically equivalent amino acids, as is known in the art and further described herein (see Table 1).
Protein-encoding DNA segments and genes of the present invention may encode a full length telomerase-associated protein or polypeptide, as may be used in expressing the protein. Such DNA segments are exemplified by those that comprise an isolated gene that includes a contiguous DNA sequence substantially as shown between position 54 and position 1799 of SEQ ID NO:29, that encodes a polypeptide substantially as shown in SEQ ID NO:16; or that includes a contiguous DNA sequence substantially as shown between position 78 and position 1094 of SEQ ID NO:30, that encodes a polypeptide substantially as shown in SEQ ID NO:18; or that includes a contiguous DNA sequence substantially as shown between position 2 and position 2368 of SEQ ID NO:19, that encodes a polypeptide substantially as shown in SEQ ID NO:20; or that includes a contiguous DNA sequence substantially as shown between position 55 and position 699 of SEQ ID NO:31, that encodes a polypeptide substantially as shown in SEQ ID NO:22; or that includes a contiguous DNA sequence substantially as shown between position 3 and position 1955 of SEQ ID NO:23, that encodes a polypeptide substantially as shown in SEQ ID NO:24.
For both protein expression and hybridization, the nucleic acid segments used may include the full length versions of any of the telomerase-associated genes disclosed herein, or their biological equivalents, including their complementary sequences where hybridization is concerned. This is exemplified by DNA segments that have, or that comprise a sequence region that has, the 1301 nucleotides of SEQ ID NO:1, the 1882 nucleotides of SEQ ID NO:29, the 1094 nucleotides of SEQ ID NO:30, the 2434 nucleotides of SEQ ID NO:19, the 807 nucleotides of SEQ ID NO:31, the 2117 nucleotides of SEQ ID NO:23, or any substantially equivalent sequences.
Further, the present DNA segments may be used to express protein fragments or peptides, for example, peptides of from about 15 to about 30, about 50 or about 100 amino acids in length. The peptides may, of course, be of any length between or around such stated ranges, with xe2x80x9caboutxe2x80x9d meaning a range of lengths in positive integers between each above-listed reference point and higher, with 12-15 or so being the minimum length. Appropriate coding sequences and regions may be readily identified from any of the regions of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:19, SEQ ID NO:31 or SEQ ID NO:23.
The sequence or coding regions of the invention will be a minimum length of 17 nucleotides, and will most often be longer than this, such as upwards of about 40-50 nucleotides in length or so. The maximum length of the DNA segments is not limited by the length of the coding regions themselves, so that DNA segments of about 1,000, about 3,000, about 5,000 and 10,000 or even longer are contemplated. It will be readily understood that all lengths intermediate between the above-quoted ranges are also included.
It will also be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5xe2x80x2 or 3xe2x80x2 sequences, and yet still be substantially as shown in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5xe2x80x2 or 3xe2x80x2 portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes.
Excepting intronic or flanking regions, and allowing for the degeneracy of the genetic code, sequences that have between about 70% and about 80%; or more preferably, between about 80% and about 90%; or even more preferably, between about 90% and about 99% of nucleotides that are identical to the nucleotides of SEQ ID NO:1, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:19, SEQ ID NO:31 or SEQ ID NO:23 will be sequences that are substantially as shown in such sequences. From the inventors"" experience, sequences with 70% identity or higher are expected to be telomerase-related sequences.
The nucleic acid segments of the present invention, regardless of the length of any coding sequences themselves, may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.
As stated above, the invention is not limited to the particular sequences of SEQ ID NO:1, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:19, SEQ ID NO:31 or SEQ ID NO:23 (nucleic acid), or SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22 or SEQ ID NO:24 (amino acid). In terms of expression, recombinant vectors may therefore variously include the telomerase-associated protein coding regions themselves, coding regions bearing selected alterations or modifications in the basic coding region, or they may encode larger polypeptides that nevertheless include such telomerase-associated protein coding regions or may encode biologically functional equivalent proteins or peptides that have variant amino acids sequences.
For protein expression embodiments, the DNA segments may include biologically functional equivalent protein-coding sequences that have arisen as a consequence of codon redundancy and functional equivalency, as is known to occur naturally within biological sequences. Alternatively, functionally equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by man may be introduced through the application of site-directed mutagenesis techniques, e.g., to introduce improvements to the antigenicity of the protein or to test telomerase mutants in order to examine their activity at the molecular level.
If desired, one may also prepare fusion proteins and peptides, e.g., where the telomerase-associated protein coding regions are aligned within the same expression unit with other proteins or peptides having desired functions, such as for purification or immunodetection purposes (e.g., proteins that may be purified by affinity chromatography and enzyme label coding regions, respectively).
Recombinant vectors form further aspects of the present invention. Particularly useful vectors are contemplated to be those vectors in which an RNA or protein coding portion of a DNA segment, whether encoding an RNA template, a full length protein or smaller peptide, is positioned under the control of a promoter. The promoter may be in the form of the promoter that is naturally linked to a telomerase-associated gene, as may be obtained by isolating the 5xe2x80x2 non-coding sequences located upstream of the coding segment or exon, for example, using recombinant cloning and/or PCR technology, in connection with the compositions disclosed herein.
In other expression embodiments, it is contemplated that certain advantages will be gained by positioning a coding DNA segment or sequence region under the control of a recombinant, or heterologous, promoter. As used herein, a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with a telomerase-associated gene in its natural environment. Such promoters may include yeast promoters normally associated with other genes, and/or promoters isolated from any other bacterial, viral, insect or mammalian cell.
Naturally, it will be important to employ a promoter that effectively directs the expression of the DNA segment in the cell type, organism, or even animal, chosen for expression. The use of promoter and cell type combinations for protein expression is generally known to those of skill in the art of molecular biology, for example, see Sambrook et al. (1989). The promoters employed may be constitutive, or inducible, and can be used under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins or peptides.
Preferred promoter systems contemplated for use in high-level expression in S. cerevisiae include, but are not limited to, the GAL1, MET3, and PGK promoter systems. For conditional alleles, as may be used in cellular studies of the RNA template, a chimeric fusion of an RNA template gene may be placed under the regulation of a heterologous promoter. Appropriate promoters include the MET3 promoter, which is repressed in the presence of methionine and induced when methionine is absent from the medium; and the GAL1,10 UAS, as described in Example XI.
B. Nucleic Acid Hybridization
In addition to their use in directing the expression of telomerase-associated RNA and protein components, the nucleic acid sequences disclosed herein also have a variety of other uses, for example, in nucleic acid hybridization embodiments. The ability of nucleic acid probes or primers to specifically hybridize to the telomerase-associated nucleic acid sequences disclosed herein will enable them to be of use in detecting the presence of complementary sequences in a given sample. However, other uses are envisioned, including the use of the sequence information for the preparation of mutant species primers, or primers for use in preparing other genetic constructs.
The present invention thus concerns nucleic acid segments of 17 nucleotides in length, or longer, that hybridize to the telomerase-associated sequences of SEQ ID NO:1, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:19, SEQ ID NO:31 or SEQ ID NO:23, or their complements, under standard hybridization conditions. This provides another physical and functional definition for identifying additional sequences in accordance with the invention, as well as defining useful sub-sequences, such as primers.
The nucleic acids that hybridize to the sequences of SEQ ID NO:1, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:19, SEQ ID NO:31 or SEQ ID NO:23, may be 17 nucleotides in length or longer, such as about 20, about 25, about 30, about 50, about 75, about 100, about 150, about 200, about 250, about 500, about 750 or about 1,000 nucleotides in length, or even longer. As the length of the nucleic acid segment that hybridizes is not solely a function of the length of the substantially complementary sequence region, these nucleic acid segments may also be about 2,000, about 3,000, about 5,000 or about 10,000 nucleotides in length or longer, so long as the total length does not prevent hybridization under the conditions defined herein.
As with the sequence or coding regions defined hereinabove, it will be readily understood that any intermediate length between the quoted ranges is included, such as 17, 18, 19, 20, 21, 22, 23, etc; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; including all positive integers through the 150-500; 500-1,000; 1,000-2,000; 2,000-5,000; and 5,000-10,000 ranges, up to and including sequences of about 12,001, 12,002, 13,001, 13,002 and the like.
The total size of nucleic acid segment or fragment, as well as the size of the complementary stretch(es), will ultimately depend on the intended use or application of the particular nucleic acid segment. The use of a hybridization probe of about 17 nucleotides in length allows the formation of a duplex molecule that is both stable and selective.
Accordingly, the nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of telomerase-associated genes or cDNAs. Depending on the application envisioned, one will desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence.
For applications requiring high selectivity, one will typically desire to employ relatively stringent conditions to form the hybrids, e.g., one will select relatively low salt and/or high temperature conditions that tolerate little, if any, mismatch between the probe and the template or target strand. Standard high stringency hybridization conditions are described in the hybridization protocols set forth herein in the detailed description.
Of course, for some applications, for example, where one desires to prepare mutants employing a mutant primer strand hybridized to an underlying template, or where one seeks to isolate telomerase-associated sequences from related species, functional equivalents, or the like, less stringent hybridization conditions are useful to allow formation of the heteroduplex. In these circumstances, one may desire to employ standard low stringency hybridization conditions, which are also described in the hybridization protocols set forth in the detailed description.
Cross-hybridizing species can thereby be readily identified as positively hybridizing signals with respect to control hybridizations. In any case, it is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex, in the same manner as increased temperature. Thus, hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results.
Where hybridization probes or primers are to be designed from a consideration of the longer sequences disclosed herein, they may be selected from any portion of any of the nucleic acid sequences. All that is required is to review the sequences and to select any continuous portion of the sequence, from 17 nucleotides in length up to and including the full length sequence.
Once the coding sequence of a telomerase-associated gene has been determined, various primers can be designed around that sequence. Primers may be of any length, but typically, are 17, 20, 25 or 30 bases or so in length. By assigning numeric values to a sequence, for example, the first residue is 1, the second residue is 2, and the like, an algorithm defining all primers is:
n to n+y
where n is an integer from 1 to the last number of the sequence and y is the length of the primer minus one, in the above cases (16, 19, 24, 29), where n+y does not exceed the last number of the sequence. For example, for the TLC1 gene, n is 1 to 1301. Thus, for a 17-mer, the probes correspond to bases 1 to 17, 2 to 18, 3 to 19 . . . up to 1285 to 1301. Table 2 herein sets forth the number of contiguous 17-mer sequences that may be obtained from the sequences of SEQ ID NO:1, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:19, SEQ ID NO:31 or SEQ ID NO:23, or their complements.
The choice of probe and primer sequences may be governed by various factors, such as, by way of example only, one may wish to employ primers from towards the termini of the total sequence, or from the ends of any functional domain-encoding sequences, in order to amplify further DNA; one may employ probes corresponding to the entire DNA, or to the RNA template region, to clone template genes from other species or to clone further telomerase template-like or homologous genes from any species including human; one may also design appropriate probes or primers to screen biological samples to identify cells with inappropriate telomerase levels or activity, as may be related to cancer or even to infertility.
The process of selecting and preparing a nucleic acid segment that includes a contiguous sequence from within SEQ ID NO:1, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:19, SEQ ID NO:31 or SEQ ID NO:23 may be readily achieved by, for example, directly synthesizing the fragment by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer. Also, fragments may be obtained by application of nucleic acid reproduction technology, such as the PCR technology of U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,682,195 (each incorporated herein by reference), by introducing selected sequences into recombinant vectors for recombinant production, and by other recombinant DNA techniques generally known to those of skill in the art of molecular biology. Of course, smaller nucleic acid fragments may also be obtained by other techniques such as, e.g., by mechanical shearing or by restriction enzyme digestion.
In certain embodiments, it will often be advantageous to employ nucleic acid sequences of the present invention in combination with an appropriate means, such as a label, for determining hybridization. A wide variety of appropriate indicator means are known in the art, including fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal. In certain embodiments, one will likely desire to employ a fluorescent label or an enzyme tag, such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmental undesirable reagents. In the case of enzyme tags, colorimetric indicator substrates are known that can be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples.
In general, it is envisioned that the hybridization probes described herein will be useful both as reagents in solution hybridization as well as in embodiments employing a solid phase. In embodiments involving a solid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to a selected matrix or surface. This fixed, single-stranded nucleic acid is then subjected to specific hybridization with selected probes under desired conditions. The selected conditions will depend on the particular circumstances based on the particular criteria required (depending, for example, on the G+C contents, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.). Following washing of the hybridized surface so as to remove nonspecifically bound probe molecules, specific hybridization is detected, or even quantified, by means of the label.
C. Further Telomerase Compositions
The present invention further includes isolated RNA segments, of from 17 to about 1,500 nucleotides in length, that comprise a non-ciliate, or preferably, a yeast telomerase RNA template. The isolated RNA segments will be obtained free from total nucleic acids, chromosomes and intact telomerase complexes, and will include a non-ciliate eukaryotic, or preferably, a yeast telomerase RNA template. This is exemplified by RNA segments including the S. cerevisiae RNA template sequence of CACCACACCCACACAC (SEQ ID NO:3).
Isolated RNA segments that include the minimum functional mammalian, drosophila or yeast telomerase RNA template coding sequences and the minimum upstream sequences necessary for expression are also contemplated. These may be identified as described herein in Example XI and will be useful in mutant analysis, promoter and expression analysis and creation of conditional mutants.
Isolated RNA segments that have substantially the same secondary structure as the RNA segment encoded by the sequence of SEQ ID NO:1 are also included within the scope of the present invention. This may be assessed by techniques, and computer programs, that predict secondary structure based on the primary sequence of the RNA. The secondary structure predictions are supported by mutant/function analysis, as is well known in the art. That is, given the predicted structure, it is straightforward for the ordinary artisan to accurately predict the effects of certain sets of mutations in the RNA.
Further compositions in accordance with this invention include affinity columns that comprise a deoxyoligonucleotide attached to a solid support, where the deoxyoligonucleotide includes a sequence complementary to a non-ciliate, or preferably, a yeast telomerase RNA template sequence. Such deoxyoligonucleotides and affinity columns will be capable of binding eukaryotic, or preferably, yeast telomerase complexes, enabling their purification. As the template RNA includes the CA-rich template region, an appropriate column-bound bait will be a GT-rich DNA sequence, as represented, by way of example only, by SEQ ID NO:2.
The oligonucleotides may be attached to any one of a variety of solid supports for use in standard column chromatography or in FPLC or HPLC techniques. Oligonucleotides may be attached using a variety of appropriate methods, such as, by way of example, using direct chemical conjugation, or other means such as biotin-avidin linkers, and the like. All such techniques are routine in the art.
Still further embodiments of the present invention concern recombinant host cells that contain or incorporate a DNA segment or recombinant vector that comprises an isolated gene associated with non-ciliate eukaryotic, or preferably, with yeast telomerase. The telomerase-associated components, whether they be cDNA or genomic, may be used in expression systems for the recombinant preparation of RNA templates or telomerase-associated polypeptides.
As used herein, the term xe2x80x9cengineeredxe2x80x9d or xe2x80x9crecombinantxe2x80x9d cell is intended to refer to a cell into which an exogenous DNA segment or gene, such as a cDNA or gene encoding a telomerase-associated component has been introduced. Therefore, engineered cells are distinguishable from naturally occurring cells that do not contain a recombinantly introduced exogenous DNA segment or gene. Engineered cells are thus cells having a gene or genes introduced through the hand of man. Recombinantly introduced genes will either be in the form of a cDNA gene (i.e., they will not contain introns), a copy of a genomic gene, or will include genes positioned adjacent to a promoter not naturally associated with the particular introduced gene.
The engineering of DNA segment(s) for expression in prokaryotic or eukaryotic systems is performed using techniques known to those of skill in the art, and further described herein in detail. It is believed that virtually any prokaryotic or eukaryotic host cell system may be employed in the expression of one or more telomerase-associated components, with yeast systems being preferred in certain embodiments. Telomerase-associated polypeptides may also be as fusions with, e.g., xcex2-galactosidase, ubiquitin, Schistosoma japonicum glutathione S-transferase, and the like.
To achieve expression, one would position the telomerase coding sequences adjacent to and under the control of a promoter. It is understood in the art that to bring a coding sequence under the control of such a promoter, one positions the 5xe2x80x2 end of the transcription initiation site of the transcriptional reading frame of the protein between about 1 and about 50 nucleotides or so xe2x80x9cdownstreamxe2x80x9d of (i.e., 3xe2x80x2 of) the chosen promoter.
Where eukaryotic expression is contemplated, one will also typically desire to incorporate into the transcriptional unit which includes the enzyme, an appropriate polyadenylation site (e.g., 5xe2x80x2-AATAAA-3xe2x80x2) if one was not contained within the original cloned segment. Typically, the poly A addition site is placed about 30 to 2000 nucleotides xe2x80x9cdownstreamxe2x80x9d of the termination site of the protein at a position prior to transcription termination.
Generally speaking, it may be more convenient to employ as the recombinant gene a cDNA version of the gene. It is believed that the use of a cDNA version will provide advantages in that the size of the gene will generally be much smaller and more readily employed to transfect the targeted cell than will a genomic gene, which will typically be up to an order of magnitude larger than the cDNA gene. However, the inventors do not exclude the possibility of employing a genomic version of a particular gene where desired.
The recombinant host cells of the invention will effectively expresses a DNA segment to produce a telomerase RNA template or a polypeptide associated with telomerase. The invention thus further includes recombinant gene products that are prepared by expressing a eukaryotic, or preferably, a yeast telomerase-associated gene in a recombinant host cell and purifying the expressed gene product away from total recombinant host cell components. The gene products include telomerase RNA templates, proteins, polypeptides and peptides associated with telomerase, and combinations and equivalents thereof.
The preparation of such recombinant gene products is preferably achieved by using a DNA segment of the invention in the preparation of a recombinant vector in which a telomerase-associated gene is positioned under the control of a promoter. The recombinant vector is then introduced into a recombinant host cell, which is cultured under conditions effective, and for a period of time sufficient, to allow expression of the telomerase-associated gene, which thus allows the expressed gene product to be collected, giving a purified preparation.
The invention further concerns recombinant RNA segments that include non-ciliate telomerase RNA templates, such as mammalian, drosophila, or preferably, yeast telomerase RNA templates; and
recombinant protein and polypeptide compositions, free from total cell components, that comprise one or more purified non-ciliate, or preferably, yeast telomerase-associated components. These are exemplified by polypeptides that include a contiguous amino acid sequence from SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22 or SEQ ID NO:24.
The terms xe2x80x9cpurified telomerase-associated polypeptide and RNA templatexe2x80x9d, as used herein, refer to telomerase-associated polypeptide or RNA template compositions, isolatable from eukaryotic, or preferably, from yeast cells, wherein the polypeptide or RNA is purified to any degree relative to its naturally-obtainable state, e.g., relative to its purity within a cellular extract. More preferably, xe2x80x9cpurifiedxe2x80x9d refers to telomerase-associated polypeptide or RNA template compositions that have been subjected to fractionation to remove various non-telomerase components. xe2x80x9cSubstantially purifiedxe2x80x9d native and recombinant telomerase RNA templates and polypeptides are also preparable using the methods of the invention.
To prepare a purified telomerase-associated component in accordance with the present invention one would subject a composition to fractionation to remove various non-telomerase-associated components. Various techniques suitable for use in RNA and protein purification will be well known to those of skill in the art. Protein purification techniques include, for example, precipitation with ammonium sulphate, PEG, antibodies, and the like, or by heat denaturation, followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques.
Specific examples of purification schemes for use in the present invention are those including initial separation of nuclear proteins, followed by gradient centrifugation methods (equilibrium and sedimentation velocity), column chromatography and gel electrophoresis, as described in Example XV. Specific binding to RNA or DNA segments related to the telomerase template sequences, including affinity column binding embodiments, is also envisioned to be particularly useful.
For assays of intact or relatively intact telomerase complexes, deoxyoligonucleotide substrates, representing 3xe2x80x2 G-rich telomere tails, are incubated in cellular extracts containing telomerase with 32P-labeled dNTP""s (typically dGTP or dTTP). The products of telomerase elongation on the input deoxyoligonucleotide substrate may then be detected by, e.g., gel electrophoresis and autoradiography. A series of substrates is also preferably used, as described in Example XV.
Although preferred for use in certain embodiments, there is no general requirement that the RNA or proteins always be provided in their most purified state. Indeed, it is contemplated that less substantially purified telomerase-associated components, which are nonetheless enriched relative to their natural state, will have utility in certain embodiments. These include, for example, certain binding assays, screening protocols, titration of components, and the like. Inactive protein fractions also have utility, for example, in antibody generation.
In further embodiments, the invention also provides polyclonal or monoclonal antibodies that bind to a non-ciliate, and preferably, to a yeast telomerase-associated polypeptide, as exemplified by an antibody that has binding affinity for a protein or peptide that includes a contiguous amino acid sequence from SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22 or SEQ ID NO:24. Cross-reactive antibodies are also encompassed by the invention, as may be identified by employing a competition binding assay, such as an ELISA or RIA, as are well known in the art.
Particular techniques for preparing antibodies in accordance with the invention are disclosed herein, which methods generally comprise administering to an animal a composition comprising an immunologically effective amount of a telomerase-associated component protein, peptide or other epitopic composition. By xe2x80x9cimmunologically effective amountxe2x80x9d is meant an amount of a telomerase-associated protein or peptide composition that is capable of generating an immune response in the recipient animal, and particularly, in this case, generating an antibody or B cell response.
Any of the DNA, RNA, proteins, polypeptides and antibodies of this invention may also be linked to a detectable label, such as a radioactive, fluorogenic, biological, chromogenic or even a nuclear magnetic spin resonance label. Biolabels such as biotin and enzymes that are capable of generating a colored product upon contact with a chromogenic substrate will be preferred in certain embodiments. Exemplary enzyme labels include alkaline phosphatase, hydrogen peroxidase, urease and glucose oxidase enzymes.
In still further embodiments, the invention concerns molecular biological and immunodetection kits. The labelled nucleic acid segments, proteins or antibodies may be employed to detect other telomerase-associated nucleic acid, protein or antibody components in extracts, cells or biological samples, as may be used in the detection of telomerase in clinical samples, or in the purification of telomerase-associated components, as appropriate. The kits will generally include a suitable telomerase-associated nucleic acid segment or antibody together with an detection reagent, and a means for containing the telomerase-associated component and reagent.
The detection reagent will typically comprise a label associated with the telomerase nucleic acid segment or antibody, or even associated with a secondary binding ligand. Exemplary ligands include secondary antibodies directed against a first antibody. The kits may contain telomerase-associated nucleic acid segments or antibodies either in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user of the kit.
Kits for use in molecular biological tests to identify telomerase-associated components may also contain one or more unrelated nucleic acid probes or primers for use as controls, and optionally, one or more further molecular biological reagents, such as restriction enzymes or PCR components. The components of the kits will preferably be packaged within distinct containers.
The container means for any of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the nucleic acid or antibody may be placed, and preferably suitably aliquoted. Where a second component, e.g., a binding ligand is provided, the kit will also generally contain a second vial or other container into which this ligand or antibody may be placed. The kits of the present invention will also typically include a means for containing the vials in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vials are retained.
D. Telomerase-Associated Methods
The invention further provides methods for detecting non-ciliate eukaryotic, and preferably, yeast telomerase-associated genes or nucleic acid segments in samples, such as cells, cellular extracts, partially purified telomerase compositions and other biological and even clinical samples. Such methods generally comprise obtaining sample nucleic acids from a sample suspected of containing a telomerase-associated gene; contacting the sample nucleic acids with a telomerase-associated nucleic acid segment as described herein under conditions effective to allow hybridization of substantially complementary nucleic acids; and detecting the hybridized complementary nucleic acids thus formed.
A variety of hybridization techniques and systems are known that can be used in connection with the telomerase detection aspects of the invention. For example, in situ hybridization, Southern blotting, Northern blotting and PCR technology. In situ hybridization describes the techniques wherein the target nucleic acids contacted with the probe sequences are located within one or more cells, such as cells within a clinical sample or cells grown in culture. As is well known in the art, the cells may be prepared for hybridization by fixation, e.g. chemical fixation, and placed in conditions that allow for the hybridization of a detectable probe with nucleic acids located within the fixed cell.
Alternatively, target nucleic acids may be separated from a cell or clinical sample prior to contact with a probe. Any of the wide variety of methods for isolating target nucleic acids may be employed, such as cesium chloride gradient centrifugation, chromatography (e.g., ion, affinity, magnetic), phenol extraction and the like. Most often, the isolated nucleic acids will be separated, e.g., by size, using electrophoretic separation, followed by immobilization onto a solid matrix, prior to contact with the labelled probe. These prior separation techniques are frequently employed in the art and are generally encompassed by the terms xe2x80x9cSouthern blottingxe2x80x9d, that detects DNA and xe2x80x9cNorthern blottingxe2x80x9d, that detects RNA. Virtually of the methods may be adapted for clinical or diagnostic assays, including diagnostic PCR technology.
In general, the xe2x80x9cdetectionxe2x80x9d of telomerase sequences is accomplished by attaching or incorporating a detectable label into the nucleic acid segment used as a probe and xe2x80x9ccontactingxe2x80x9d a sample with the labeled probe. In such processes, an effective amount of a nucleic acid segment that comprises a detectable label (a probe), is brought into direct juxtaposition with a composition containing target nucleic acids. Hybridized nucleic acid complexes may then be identified by detecting the presence of the label, for example, by detecting a radio, enzymatic, fluorescent, or chemiluminescent label.
These detection methods may be employed to detect telomerase-associated genes, whether RNA- or protein-encoding, in both clinical and laboratory samples, e.g., as may be used in telomerase purification, analysis, mutagenesis and the like. In cells or cellular extracts obtained from an animal or human patient, the detection of telomerase may have particular relevance, for example, in the diagnosis or detection of tumor cells within a sample suspected of containing such cells. This is supported by recent findings linking telomerase to oncogenesis and various late stage tumors and tumor cells (Harley et al., 1992; Counter et al., 1992, 1994a; Shay et al., 1993; Klingelhutz et al., 1994; de Lange, 1994; Greider, 1994). The differential detection and diagnosis of malignant tumors as opposed to benign tumors is also contemplated.
Further clinical samples that may be analyzed for the presence of telomerase-associated genes, as described above, include those suspected of containing a pathogen. As telomerase activity is only present in dividing cells, testing a sample of somatic cells of an animal or human for the presence of telomerase may indicate the presence of an invading unicellular organism within the sample. This may allow disease diagnosis alone, or in combination with other methods. The diagnosis of yeast infections, for example, is an immediate application of the present invention. The development of species-specific markers for other opportunistic infections is also contemplated.
Diagnostic methods for identifying various conditions associated with infertility in animals and humans are also provided by the invention. For example, as telomerase activity is required in germ cells, including human sperm and ova, testing samples from animals and humans suspected of having a condition connected with reproductive failure would provide useful information. A negative test would likely indicate a defect in the reproductive capacity of sperm or egg cells within a given sample.
In further embodiments, the invention concerns methods based upon suppression of xe2x80x9ctelomeric silencingxe2x80x9d for use in identifying non-ciliate, and preferably, yeast telomerase-associated genes or active fragments thereof. Such methods generally comprise, initially, preparing a cell containing a chromosome that contains a genetic marker located proximal to a telomere, wherein the telomere represses the expression of the marker. Next, one would contact the cell with a composition comprising a candidate gene and identify any gene, or portion thereof, that allows expression of the marker. xe2x80x9cGenesxe2x80x9d identified in this way may be wild type genes or fragments that may disrupt the telomere function due to over-expression, or they may be mutant or truncated genes that simply do not function correctly.
Appropriate cells for use in such assays include those cells that contain an active telomere, such as eukaryotic cells that are capable of dividing, as exemplified by yeast cells, drosophila cells, and certain human cells, such as sperm, egg and cancer cells. The novel technology developed by the inventors is contemplated for use in any organism in which the telomeres cause a transcriptional repression (silencing) of nearby genes. For ease of operation, yeast and Drosophila melanogaster (fruit flies) are currently preferred. However, the use of human cells is also contemplated.
The genetic markers that are added in the vicinity of a telomere may be any marker gene that gives a readily identifiable phenotype upon expression. Such markers are also often termed xe2x80x9creporter genesxe2x80x9d. Generally, the marker or reporter genes encode a polypeptide not otherwise produced by the cell which is detectable by analysis, e.g., by visual inspection or by fluorometric, radioisotopic or spectrophotometric analysis. One example is E. coli beta-galactosidase, which produces a color change upon cleavage of an indigogenic substrate; a further example is the enzyme chloramphenical acetyltransferase (CAT), which may be employed with a variety of substrates that give detectable products; and still further examples are firefly and bacterial luciferases.
Still further marker genes for use herewith are those capable of transforming the host cell to express unique cell surface antigens, e.g., viral env proteins such as HIV gp120 or herpes gD, which are readily detectable by immunoassays. The polypeptide products of this type of marker gene are secreted, membrane bound polypeptides, or polypeptides adapted to be membrane targeted, allowing ready detection by antibodies. However, antigenic reporters are not currently preferred because, unlike enzymes, they are not catalytic and thus do not amplify their signals.
Yeast markers, when expressed, may result in a colored phenotype or result in a specific nutrient independence (prototrophy), or even in a nutrient requirement, or such like. Exemplary genetic markers that may be used in yeast include genes that are required for the biosynthesis of specific amino acids, such as HIS3, TRP1, LYS2, and LEU2. Genes that confer sensitivity to drugs, such as the CAN1 gene that confers sensitivity to canavinine are also contemplated for use. Currently preferred marker genes for use in yeast are ADE2 and URA3.
Many suitable genetic markers are also available for use in human cell systems. These include the markers based upon color detection or antigen detection, as above, and also marker genes that encode polypeptides, generally enzymes, that render the host cells resistant against toxins. These include the neo gene that protects host cells against toxic levels of the antibiotic G418; the dihydrofolate reductase genes that confer resistance to methotrexate; and the HSV tk gene that is used in conjunction with ganciclovir. Currently preferred examples are the markers neo and hprt, which are routinely used in the art.
The cells used in such assays may contain two distinct genetic markers, and each genetic marker may be located on a distinct chromosome if desired. The combined use of ADE2 and URA3 in yeast cells is currently a particularly preferred system.
As described hereinabove, human telomerase RNA template and polypeptide-encoding genes that have substantial sequence homology to the yeast sequences throughout, or in certain sequence regions, may be isolated by nucleic acid hybridization, i.e., standard cloning techniques (Sambrook et. al., 1989). However, even if the human sequences are not directly homologous, RNA template and other telomerase genes may still be isolated using the advantageous methods disclosed herein.
One suitable method for identifying a human telomerase-associated gene, is to apply the suppression of telomeric silencing protocol to a human nucleic acid library using a yeast cell system. Such methods generally comprise preparing a yeast cell containing a chromosome that contains a genetic marker located proximal to a telomere, where the telomere represses the expression of the marker; contacting the cell with a composition comprising a candidate human gene; and identifying a human gene that allows expression of the marker.
Further suitable methods for identifying human telomerase-associated genes are those based entirely upon human cells, which methods presuppose the lowest level of homology between the yeast and human cell systems. These methods comprise preparing a human cell that contains a chromosome having a genetic marker located proximal to a telomere, where the telomere represses the expression of the marker; contacting the cell with a composition comprising a candidate human gene; and identifying a human gene that allows expression of the marker.
Another method for isolating genes that encode products that interact with telomerase RNA is that which assays for genes that re-establish telomeric silencing when the template RNA is overexpressed, as described in Example XIV. Here, initially the RNA template is presumed to interact with a limiting telomerase component to form a non-functional complex. Increasing the concentration of a limiting component, by over-expression, thus re-establishes telomeric silencing. Preferably, RNA template levels that are minimally suppressive are used.
Still more approaches for identifying components that interact with telomerase RNA are described in Example XIV, which are based upon isolating mutations that enhance or suppress the phenotypes of conditional telomerase template alleles.
Further elements of this invention are non-ciliate eukaryotic, and preferably, yeast genes that are identified by any of the foregoing methods. One such gene is disclosed herein, termed TLC1, that encodes a telomerase RNA template. Other such genes are also disclosed herein, termed STR genes, that encode telomerase-associated polypeptides. Particular examples of such genes of the invention are thus TLC1, STR1, STR3, STR4, STR5 and STR6, and other non-ciliate eukaryotic, and preferably, yeast nucleic acid segments that have the physical and functional characteristics of any of the foregoing genes.
Active fragments of genes and RNA components, such as TLC1 RNA, may also be identified using the present methods. Titration assays based upon those used for the original identification of TLC1 may be used to define the minimum functional region. It is contemplated that relatively small regions of the RNA (about 50 bp) that suppress silencing will be identified. Conditional mutations made in regions of the RNA that are evolutionarily conserved, or that may interact with a limiting factor, as suggested by the titration analysis, will identify functionally important region of the telomerase RNA. Active regions of telomerase genes and RNA components may also be identified using methods for dissecting small nuclear RNAs (snRNAs), as described in Example XIII.
In still further embodiments, the invention provides methods for use in identifying candidate substances that bind to yeast and other non-ciliate eukaryotic telomerase components. These methods generally include preparing an isolated telomerase component; contacting the isolated telomerase component with a composition comprising a candidate substance under conditions effective and for a period of time sufficient to allow binding; and detecting the presence of a telomerase component-candidate substance bound complex.
It will be understood that such methods are similar in principle to the nucleic acid hybridization methods described hereinabove. Indeed, the xe2x80x9ccandidate substancesxe2x80x9d to be detected may be nucleic acids, including human nucleic acid segments, that are detected by binding to eukaryotic, and preferably, to yeast telomerase RNA or DNA components, and preferably to a defined small functional region of the template that suppress silencing, under the high or low hybridization conditions described above. However, other components that bind to telomerase may be identified by binding to the isolated RNA, DNA or polypeptide components of the present invention. These components may include proteins, polypeptides, peptides, antibodies, small molecules, cofactors and the like.
Accordingly, the present invention provides binding assays, including high throughput binding assays using recombinant expression products, for use in identifying compounds capable of binding to telomerase or to a telomerase-associated component. The binding assays will preferably use the smaller RNA fragments identified in titration or other functional assays described herein.
Further methods for identifying compounds that bind to telomerase-associated components include those based upon cellular assays. One method for identifying a candidate substance that modifies telomerase activity comprises the following steps:
preparing a eukaryotic, or preferably, a yeast cell containing a chromosome that contains a genetic marker located near to, or in the vicinity of, a telomere, the telomere capable of repressing the expression of the marker;
contacting the cell with a composition comprising a candidate substance; and
identifying a candidate substance that allows expression of the marker or that further represses the expression of the marker.
This method is most suitable for identifying candidate inhibitory substances that allow expression of the marker. However, it can also be used to identify candidate stimulatory substances that allow further repression of the marker.
To identify a compound that inhibits telomerase activity, one generally prepares a cell with a genetic marker that is substantially repressed by the telomere. Here, the marker gene is located proximal, i.e., immediately adjacent, to the telomere. Substantial repression is defined by repression to at least about 50%, or preferably, to about 25%, 10% or about 1%. However, the expression of the marker may be repressed to even about 0.01%. The inhibitory substance is then detected by detecting greater expression of the marker.
To identify a compound that activates telomerase activity, one would generally prepare a cell with a genetic marker that is either not repressed at all or that is not substantially or maximally repressed. One would then select a candidate activator by identifying a substance that establishes or allows repression or more substantial repression. This is based upon the concept that stimulating telomerase to synthesize longer than normal telomeres will result in an increase in silencing of a marker gene. To detect the increase requires that a system initially be established in which the marker gene is only minimally repressed, or even not repressed at all. This is readily achieved by inserting the marker gene in the location or vicinity of the telomere, but further away from the telomere rather than immediately adjacent to it. An increase in repression, i.e., a decrease in marker gene expression, indicates a positive candidate substance.
Still further methods for identifying compounds that functionally interact with telomerase or telomerase-associated components are those based upon the telomerase xe2x80x9chealing of broken chromosomesxe2x80x9d assay described herein. This method is conducted as generally described in Example XII and FIG. 8, using a modified Haber-based assay (Kramer and Haber, 1993). Other useful telomerase functional assays are those that analyze telomere length and cell viability with increased age of a culture (Lundblad and Blackburn, 1989), and those in vitro systems described herein based on the addition of labelled nucleotides to a telomeric-like sequence.
Any of the cellular or activity-based telomerase assays may be adapted to screen for candidate substances that modify telomerase activity. To achieve this, one would first conduct the assay in the absence of the test candidate substance to obtain an activity value in its absence. One would then add the candidate substance to the telomerase composition or cell and conduct the assay under the same conditions. Candidate substances that reduce or promote telomerase activity can thus be readily identified.
Useful telomerase-modifying compounds are not believed to be limited in any way to protein or peptidyl compounds or oligonucleotides. In fact, it may prove to be the case that the most useful pharmacological compounds identified through application of a screening assay will be non-peptidyl in nature. Accordingly, in such screening assays, it is proposed that compounds isolated from natural sources, such as animals, bacteria, fungi, plant sources, including leaves and bark, and marine samples, may be assayed for the presence of potentially useful pharmaceutical agents. It will be understood that the pharmaceutical agents to be screened could also be derived from chemical compositions or man-made compounds.
The invention thus further encompasses components that bind to telomerase and that are capable of modifying telomerase activity, as may be identified by any of the foregoing binding and/or functional or cellular assay methods. This results in compositions of telomerase activators or inhibitors, including pharmaceutically acceptable compositions, and methods for modifying telomerase activity.
In yet still further embodiments, the present invention thus also provides methods for modifying the replicative capacity of a cell, which methods comprise contacting a telomerase-containing cell with an amount of a component or substance effective to modify telomerase activity. xe2x80x9cModifyingxe2x80x9d in this context includes both compositions and methods for inhibiting telomerase activity, as may be used, e.g., in inhibiting or killing a tumor cell or a pathogen; and compositions and methods for stimulating telomerase activity, as may be used in embodiments connected with promoting the replication of a cell, such as in treating infertility.
Where the telomerase-containing cells are located within an animal, a pharmaceutically acceptable composition of the telomerase activator or inhibitor may be administered to the animal in an amount effective to modify the telomerase activity of the target cell. In terms of inhibiting telomerase activity in tumor cells, this is contemplated to be an effective mechanism by which to treat cancer that will have very limited side effects. Similarly, effective antimicrobial treatments are contemplated, as are applications in treating age-related disorders such as atherosclerosis and osteoporosis. Further, gene therapy using functional telomerase-associated genes is envisioned to be of use in treating telomerase dysfunction, as could provide a treatment for infertility in humans and other animals.